Under auspices of this grant, my colleagues and I have shown that PDGF AA and the PDGF alpha receptor are expressed in pregastrula embryos of both mice and frogs. We identified an embryonic lethal mouse mutant "patch" wherein coding sequence for the PDGF alpha receptor are deleted. We and others showed that anatomical defects in patch homozygotes track with expression of the PDGF alpha receptor in wild type mouse embryos. In the face of these observations, the link between PDGF and early embryonic development must still be considered tenuous. There is no evidence that PDGF alpha receptor subunits are ever activated by ligand in utero. Moreover, there is no evidence that alpha receptor activation is necessary for proper development. The patch deletion spans the entire alpha receptor gene and the boundaries of the lesion have not yet been mapped. The aim of this research is to critically evaluate the role of PDGF AA and the alpha receptor in early embryonic development. We have two broad objectives. The first objective is to determine when and where alpha receptors are actually activated in the developing mouse embryo. We have recently shown that synthetic tyrosine phosphopeptides which duplicate "informative" autophosphorylation domains can be used to raise activation state-specific antibodies to specific growth factor receptors. We will raise such antibodies to the PDGF alpha receptor and use them to display receptor activation in embryonic tissue sections. If activation of alpha receptors is an essential morphogenic event, we would expect to see activation in the tissues (and at tahe times) where failures occur in the developing Patch homozygotes. The second objective is to express a dominant negative mutation specific for the PDGF A gene product in transgenic mice. If the patch mouse syndrome does indeed reflect a loss of function mutation in the alpha receptor than transgenic mouse strains which fail to produce PDGF AA should exhibit much the same phenotype. Since expression of these mutations may be embryonic lethal, we will use a binary system for regulating transgene expression developed recently by Leder and his colleagues at Harvard medical School. In this system,. the GAL 4/UAS transcription control element of yeast is used to maintain potentially lethal transgenes in a transcriptionally silent state. Expression is triggered when these transgenic "carrier mice" are mated to a transgenic "activator mice" that expresses the yeast GAL 4 gene product under tissue-specific promoters of interest.